PROJECT SUMMARY: Fully functional mitochondria are critically important for cells, and particularly important for tissues with high-energy demands such as muscle, neurons, and the developing embryo. As such, mitochondrial dysfunction is often associated with disease, such as neurodegeneration, cardiovascular disease and diabetes. Mitochondria work in concert with the nucleus and must import hundreds of nucleus- encoded products to supplement their own small genome. We have developed Drosophila as a model system to study mitochondria and our long-term goal is to understand the molecular mechanisms that control mitochondrial function during tissue homeostasis and development. We have found the Drosophila protein Clueless (Clu) is required to support mitochondrial function. Clu is highly conserved from yeast to human and Clu family members are ribonucleoproteins that preferentially bind nucleus-encoded mRNAs destined for mitochondria. Clu forms dynamic, large, mitochondria-associated particles in the cytoplasm. Our broad objectives for this proposal are to determine how particles form and what role they play in supporting mitochondrial function and protein import. Drosophila clu mutants are sick and sterile with damaged mitochondria. Clu associates with proteins located in the mitochondrial outer membrane, including the translocase responsible for protein import. Clu also associates with the ribosome at the outer membrane. We hypothesize that Clu particle formation is important for regulating mRNA localization and protein import. To test this hypothesis, our first Aim defines Clu particle dynamics and the signals that regulates it using microscopy, genetics and molecular biology. Our second Aim examines how Clu particles contribute to mRNA regulation using microscopy, molecular biology and biochemistry. Our third Aim uses molecular biology, biochemistry and genetics to examine how Clu particles regulate mitochondrial protein levels. The knowledge we expect to gain from this proposal on Clu particle regulation will be a transformative step forward because so little is known about the ribonucleoproteins involved in mitochondrial protein import, and Clu particles represent an undefined, novel cytoplasmic particle critical for mitochondrial function. Knowledge gained from the research proposed here will significantly advance our understanding of how mitochondria-localized mRNAs are regulated, translated and imported, which is a basic, fundamental process important for mitochondrial function in all cells.